Agonists and antagonists of gonadotropin-releasing hormone-2, and use thereof

ABSTRACT

The present invention relates to agonists and antagonists of gonadotropin-releasing hormone-2 (GnRH-2), which regulates the activity of GnRH-2 by specifically binding to GnRH-2 receptors, and uses thereof. The pharmaceutical compositions comprising the GnRH-2 agonists and antagonists according to the present invention are useful for the treatment of reproductive physiology diseases and steroid-related cancer cells because they specifically bind to the GnRH-2 receptors, and they are also usefully applicable to the raising industry of non-mammalian animals, i.e., birds and fish.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates to agonists and antagonists ofgonadotropin-releasing hormone-2 (GnRH-2) which regulate the activity ofGnRH-2 by specifically binding to GnRH-2 receptors, and pharmaceuticalcompositions for regulating the GnRH-2 receptors.

(b) Description of the Related Art

Gonadotropin-releasing hormone (GnRH) is a neuropeptide having animportant role in reproductive physiology, and it is widely used as atreatment agent for endocrinal and neuroendocrinal diseases (Sealfon SC, et al., Endocr. Rev. vol.18 pp. 180-205, 1997). Thus far, about 14forms of GnRH have been found in 70 different species of vertebrates andinvertebrates, and two or more GnRHs exist in each individual (Fernald RD, et al., Front Neuroendocrinol, vol.20, pp. 224-240, 1999).

The first type of GnRH (GnRH-1) is a hypothalamic GnRH. The neurons ofGnRH-1 are mostly distributed in the hypothalamus, and their nerveterminals exist at the median eminence. GnRH-1 is secreted at the medianeminence, arrives at the pituitary gland via a portal system thatconnects the hypothalamus and pituitary gland, and regulates thefunctions of the pituitary gland. GnRH-1 regulates the secretion offollicle stimulating hormone (FSH) and luteinizing hormone (LH), and LHand FSH regulate gonadal development and steroid synthesis. Therefore,GnRH-1 is deeply involved in the regulation of reproductiveendocrinology in vertebrates (Seeburg P H, et al., Recent Prog Horm Resvol.43 pp. 69-98, 1986).

The typical GnRH-1 is mammalian GnRH (mGnRH) that was found for thefirst time in the mammalian hypothalamus (Matsuo H, et al., BiochemBiophys Res Commun vol.43 pp. 1334-1339, 1971; Burgus R, et al., ProcNatl Acad Sci USA vol.69 pp. 278-282, 1972). mGnRH is found in mostvertebrates including Aves, Reptilia, and Amphibia, as well as Mammalia.However, in fish several birds, and amphibians, different forms thereofare found (Miyamoto K, et al., Life Sci vol. 32, pp. 1341-1347, 1983;Powell J F et al., Proc Natl Acad Sci USA vol. 91 pp. 12081-12085, 1994;Yoo M S, et al., Mol Cell Endocrinol vol.164 pp. 197-204, 2000).

The second type of. GnRH (GnRH-2) is chicken GnRH-II (His⁵Trp⁷Tyr⁸GnRH,cGnRH-II) that was found for the first time in the chick brain (MiyamotoK et al, Proc Natl Acad Sci USA vol.81 pp. 3874-3878). The neurons ofcGnRH-II are mostly found in the midbrain, and their nerve terminalsexist in the posterior hypothalamus or hindbrain. Although the exactfunctions of cGnRH-II are not well known, it is postulated that itgenerally has an important role in functioning as a neuromodulator andin regulating sexual behaviors (Troskie B, et al., Neuroendocrinologyvol.65 pp. 396-402, 1997). Further, as several nerve terminals extend tothe hypothalamus, it is probably involved the regulation of reproductivephysiology. cGnRH-II is found in almost all vertebrates, i.e., Pisces,Amphibia, and Reptilia, as well as Mammalia including humans, and mutantforms thereof have not been found (White R B, et al., Proc Natl Acad SciUSA vol.95 pp. 305-309, 1998).

Hence, in vertebrates higher than Pisces, GnRHs that exist in a singlespecies are cGnRH-II (GnRH-2) and mGnRH (or a mutant form of GnRH-1).Furthermore, GnRH-1 and GnRH-2 have been known to be expressed inperipheral tissues of the body, in addition to the central nervoussystem. Particularly, the expression of GnRH-1 and GnRH-2 has beenreported in tissues regulating the immune and reproductive systems (KangS K, et al., Endocrinology vol.142 pp. 182-192, 2001; Wilson T M, etal., Mol Endocrinol vol.9 pp. 44-53, 1995; Dong K W, et al., Mol CellEndocrinol vol.117 pp. 121-130, 1996). Therefore, GnRH is expected tofunction as a local regulator in immune and reproductive systems, inaddition to its functions in the neuroendocrine regulation.

Studies of GnRH have been vigorously conducted, as they provideessential information on the design of clinical treatment agents as wellas the understanding of reproductive functions. GnRH is secreted in apulsatile manner, and such a secretion manner plays an important role inregulating the synthesis and secretion of steroid hormones in the gonadsand maintaining normal reproductive functions. Endocrine orneuroendocrine diseases generated by the modification of GnRH genes anddysfunction of GnRH neurons can be cured by treating GnRH in a pulsatilemanner. However, if GnRH is treated continuously at a highconcentration, the function of GnRH receptors becomes deficient, andultimately hypofunction of the gonads is induced. By virtue of such GnRHfunctions, GnRH is used for the treatment of reproductive endocrinediseases and precocious puberty, as well as the control of menstruationperiods in in vitro fertilization (Huirne J A, et al., Lancet vol.358pp. 1793-1803, 2001). Also, GnRH is used for the treatment of cancersthat are sensitive to steroid hormones, i.e., prostate cancer, breastcancer, and ovarian cancer (Schally A V, Peptides vol.20 pp. 1247-1262,1999; Grundker C, et al., Eur J Endocrinol vol.146 pp. 1-14, 2002).

Recent studies have revealed that GnRH-2 is more effective than GnRH-1for the treatment of some cancer cells, and that signal transduction viaGnRH-2 is different from that of GnRH-1 (Kang et al., ibid.; Grundker etal., ibid.). Also, as a receptor sensitive to GnRH-2 was identified inmonkeys (Neill J D, et al., Biochem Biophys Res Commun vol. 282 pp.1012-1018, 2001; Millar R, et al., Proc Natl Acad Sci USA vol. 98 pp.9636-9641, 2001), studies about the physiological functions of GnRH-2via such receptor became necessary. Due to the clinical importance ofGnRH, approximately 3000 GnRH agonists and antagonists have beendeveloped thus far. Most GnRH agonists and antagonists were developed bypartially modifying the amino acid structure of GnRH-1 to increase theirbinding affinity toward the GnRH receptors and to slow down their invivo degradation rate, thereby maximizing their efficiency (Sealfon etal., ibid.).

Recently, the inventors cloned three types of receptors that are verysensitive to GnRH-2 in bullfrogs (Wang et al., Proc Natl Acad Sci USAvol. 98 pp. 361-366, 2001). All of the bullfrog GnRH receptors (bfGnRHR)react more sensitively to GnRH-2 than GnRH-1. Also, the bfGnRHR showsgreat structural differences from the GnRH receptors that were found inmammals, but it is very similar in structural aspects to the GnRHreceptors found in nonmammals, i.e., Pisces, Amphibia, and Aves. Inaddition, it is structurally similar to the receptor sensitive to GnRH-2that has been recently found in monkeys (monkey GnRHR-2) (Neill et al.,ibid.; Millar et al., ibid.). Accordingly, the bfGnRHR is very similarto the second GnRH receptor of mammals in respect of its structure andfunction.

Thousands of GnRH-1 agonists have been developed because of thereproductive and physiological importance of GnRH-1, as well as itsclinical efficacy in reproductive dysfunction and cancer treatment.However, little development and study of GnRH-2 analogs has beenconducted, for several reasons. GnRH-2 was characterized relativelylater than GnRH-1, and the fact that GnRH-2 is present in mostvertebrates including humans was revealed only four years ago. Inaddition, in vivo functions of GnRH-2 have not been fully known, and thediscovery of the GnRH-2 receptor which is the most important factor inthe development of GnRH-2 analogs was delayed. In nonmammalian animals,the GnRH-2 receptors were found in catfish in 1997 and goldfish in 1998(Tensen et al., 1997; Illing et al., 1999), and recently three types ofGnRH receptors were found in bullfrogs by the inventors, and therebystudies on them became serious (Wang et al., ibid.). Moreover, in thecase of mammals, the fact that the second receptor that is sensitive toGnRH-2 is present in monkeys was found extremely recently (Millar etal., ibid.; Neill et al., ibid.]. Hence, studies regarding thedevelopment of GnRH-2 analogs have been delayed.

Several GnRH-2 agonists were developed through the substitution of the6^(th) amino acid of GnRH-2 ([D-Arg⁶]GnRH-2, [D-Leu⁶]GnRH-2,[D-Trp⁶]GnRH-2, [D-t-bu-Ser⁶]GnRH-2, Siler-Khodr and Khodr, et al.,ibid. and PCT Laid-Open Publication WO No. 01-74377). However, theseagonists studies were carried out in cell lines having receptorssensitive to GnRH-1, and they were not conducted with regard toreceptors sensitive to GnRH-2.

Therefore, the current circumstance is that the development of GnRH-1antagonists and studies about GnRH-2 receptors have been carried out,but the development of GnRH-2 agonists and antagonists has not beenconducted. Hence, for clinical applications and studies of physiologicalfunctions via GnRH-2, the development of GnRH-2 agonists and antagonistsis required.

SUMMARY OF THE INVENTION

In order to achieve the objects as mentioned above, it is an object ofthe present invention to provide agonists of gonadotropin-releasinghormone-2 (GnRH-2) which regulate the activity of GnRH-2 by specificallybinding to GnRH-2 receptors.

It is another object of the invention to provide antagonists ofgonadotropin-releasing hormone-2 (GnRH-2) which inhibit the activity ofGnRH-2 by specifically binding to GnRH-2 receptors.

It is a further object of the invention to provide pharmaceuticalcompositions for regulating the release of gonadotropin comprising theGnRH-2 antagonists or agonists as active ingredients.

In order to achieve the objects as mentioned above, the presentinvention relates to agonists of gonadotropin-releasing hormone-2(GnRH-2) which regulate the activity of GnRH-2 by specifically bindingto GnRH-2 receptors.

Also, the invention relates to a pharmaceutical composition forregulating the release of gonadotropin comprising the GnRH-2 agonists asan active ingredient, and pharmaceutically acceptable carriers.

Also, the invention relates to antagonists of gonadotropin-releasinghormone-2 (GnRH-2) which regulate the activity of GnRH-2 by specificallybinding to GnRH-2 receptors.

Also, the invention relates to a pharmaceutical composition forregulating the release of gonadotropin comprising the GnRH-2 antagonistsas an active ingredient, and pharmaceutically acceptable carriers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequences of GnRH-1, GnRH-2, and their analogs (SEQ IDNOs: 1-13 and 15).

FIG. 2 a to FIG. 2 d are graphs showing the inositol phosphateproduction after treating GnRH-1, GnRH-2, and GnRH-2 agonists to eachcell line expressing the GnRH-2 receptors, i.e., bfGnRHR-1, bfGnRHR-2,and bfGnRHR-3, and the GnRH receptor of rats.

FIG. 3 a to FIG. 3 b show luciferase activity after treating GnRH-2 andGnRH-2 agonists to cells expressing the GnRH-2 receptor that was foundin Primates.

FIG. 4 shows the concentration of serum LH (luteinizing hormone) andtestosterone over the lapse of time after treating 10 μg of a GnRH-2agonist ([D-Ala⁶]GnRH-2) into a rat.

FIG. 5 a to FIG. 5 c show that a receptor selectively binding to GnRH-2is present in TSU-Pri cells, human prostate cancer cells.

FIG. 6 a to FIG. 6 c are graphs showing the inositol phosphateproduction after treating Trptorelix-1 or Cetrorelix in the presence of10 nM GnRH-2 to GH3 each cell line expressing the GnRH-2 receptors,i.e., bfGnRHR-2 and bfGnRHR-3, and rat GnRH receptor.

FIG. 7 is a graph showing the inhibition of GnRH-2-induced luciferaseactivity by treating Trptorelix-1, -2, and -3, which are GnRH-2antagonists, and Cetrorelix, a GnRH-1 antagonist to cells expressing theGnRH-2 receptor that was found in Primates.

FIG. 8 a and 8 b are graphs showing binding abilities between GnRH-1,Cetrorelix, GnRH-2, Trptorelix-1, and GnRH-2 agonists, and receptors ineach cell line expressing bullfrog GnRH-2 receptor and rat GnRHreceptor.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The inventors recently identified three types of GnRH receptors(bfGnRHR) that are very sensitive to GnRH-2 in bullfrogs for the firsttime (Wang et al., ibid), and they recently cloned a receptor that issensitive to GnRH-2 in the genome of cells in Primates. The amino acidsequence of this receptor differs by two amino acids from that of thePrimate GnRH-2 receptor that has already been characterized (Neill etal., ibid.). By using them, the inventors constructed cell linesexpressing rat GnRH receptors, which very sensitively react to GnRH-1,and bfGnRHR, which sensitively reacts to GnRH-2, by theretrovirus-mediated infection method, and investigated the activities ofthe receptors by treating them with GnRH-2 agonists or antagonists.Also, cells expressing the GnRH-2 receptor that was found in Primateswere treated with GnRH-2 and its agonists to determine the luciferaseactivities. As a result, it was revealed that the inositol phosphateproduction and the luciferase activity were increased depending on theconcentration of these agonists and antagonists. [D-Ala⁶]GnRH-2 hadhigher specific sensitivity than a wild type ligand; and the inventorsfound that Trptorelix-1 and Trptorelix-2, which are antagonists ofGnRH-2, show very high sensitivity as compared with Cetrorelix, whichwas already known for a GnRH-1 antagonist, and that they inhibit theactivity of GnRH-2 receptors, and thus completed the present invention.

Hereafter, the present invention will be described in more detail.

The present invention provides agonists of GnRH-2 that regulate theactivity of GnRH-2 by specifically binding to GnRH-2 receptors. Theamino acid sequences of GnRH-1 and GnRH-2 in mammals including humansare shown below and in FIG. 1. The amino acid sequences of GnRH-1 andGnRH-2 show differences at the 5^(th), 7^(th), and 8^(th) amino acids.

GnRH-1: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH₂ (SEQ ID NO: 13).

GnRH-2 (SEQ ID NO: 1): pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-GlyNH₂

Preferably, the GnRH-2 agonists according to the present invention arepeptides where the 6^(th) amino acid of the GnRH-2 is D-Ala or D-Lys,and more preferably they have a peptide sequence (SEQ ID NO: 2) as shownbelow. [D-Lys⁶]GnRH-2 shown in SEQ ID NO: 4 is a peptide In which D-Alais modified into D-Lys at the 6^(th) amino acid, and all other sequencesshown in SEQ ID NO: 3 and SEQ ID NOS: 5 to 8 are those whose 6^(th)amino acid is substituted with D-Ala.

pyroGlu-His-Trp-Ser-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Pro-GlyNH₂

In the above peptide sequence,

Xaa₅ is Tyr, His, or Leu;

Xaa₆ is D-Ala, or D-Lys;

Xaa₇ is Leu, Tyr, or Trp; and

Xaa₈ is Leu, Tyr, or Trp.

Most preferably, the GnRH-2 agonists are selected from the groupconsisting of the peptides shown in SEQ ID NO: 3 to SEQ ID NO: 8, andthese sequences are illustrated below. In the amino acid sequences ofGnRH-2 agonists shown below, amino acids that are different from theamino acid sequence of GnRH-2 are represented in bold Italics.

[D-Ala⁶]GnRH-2(SEQ NO: 3):p-Glu-His-Trp-Ser-His-D-Ala-Trp-Tyr-Pro-GlyNH₂

[D-Lys⁶]GnRH-2(SEQ ID NO: 4):p-Glu-His-Trp-Ser-His-D-Lys-Trp-Tyr-Pro-Gly-NH₂

[D-Ala⁶, Leu⁷]GnRH-2(SEQ ID NO: 5):pGlu-His-Trp-Ser-His-D-Ala-Leu-Tyr-Pro-Gly-NH₂

[Leu⁵, D-Ala⁶]GnRH-2(SEQ ID NO: 6):pGlu-His-Trp-Ser-Leu-D-Ala-Trp-Tyr-Pro-Gly-NH₂

[Tyr⁵, D-Ala⁶, Leu⁶]GnRH-2(SEQ ID NO: 7):pGlu-His-Trp-Ser-Tyr-D-Ala-Trp-Leu-Pro-Gly-NH₂

[D-Ala6, Tyr⁷, Trp⁸]GnRH-2(SEQ ID NO: 8):pGlu-His-Trp-Ser-His-D-Ala-Tyr-Trp-Pro-Gly-NH₂.

[Tyr⁵, D-Ala⁶, Leu⁸]GnRH-2 is a peptide in which the 6^(th) amino acidin salmon GnRH, which is GnRH-3, is modified into D-Ala; Trp at the7^(th) position is that of GnRH-2; the 5^(th) amino acid is Tyr that isfound in GnRH-1; and the 8^(th) amino acid is Leu of GnRH-3. [D-Ala⁶,Tyr⁷, Trp⁸]GnRH-2 is an analog in which the 7^(th) and 8^(th) aminoacids of GnRH-2 are substituted with each other, while [Leu⁵,D-Ala⁶]GnRH-2 is a GnRH-2 analog whose 5^(th) amino acid is substitutedwith Leu.

Gly, the 6^(th) amino acid of GnRH-2, is a position on which abeta-II-turn is formed when the GnRH binds to its receptor. Thesubstitution of Gly with D-Ala contributes to a stable beta-II-turnconformation of GnRH. In the case of [D-Trp⁶]GnRH-2 where said Gly issubstituted with D-Trp, it shows a binding affinity and sensitivitysimilar to those of GnRH-2, whereas [D-Ala⁶]GnRH-2 shows a much higherbinding affinity and sensitivity than GnRH-2. Consequently, D-Alasubstitution is regarded to increase the interaction with the receptors.

Also, the present invention relates to GnRH-2 (gonadotropin-releasinghormone-2) antagonists which inhibit the activity of GnRH-2 byspecifically binding to GnRH receptors, and the GnRH-2 antagonists arepreferably GnRH-2 antagonists comprising the following peptide sequence(SEQ ID NO: 9).

Ac-D-2Nal-(A)-D-Phe-D-3Pal-Ser-Xaa₅-D-Cit-Trp-Xaa₈-Pro-D-AlaNH₂

In the above peptide sequence,

A is 4Cl, 4F, or 4Br, and preferably 4Cl;

Xaa₅ is Tyr or His; and

Xaa₈ is Tyr or Leu.

The above-modified amino acids are compound names that are widely usedin the art to which the invention pertains, and specifically, Ac-D-Nalrefers to D-alanine which is substituted with a naphthyl on thebeta-carbon atom and is further substituted with an acetyl group(β-(2-naphthyl-D-Ala)); D-3Pal refers to D-alanine which is substitutedwith a pyridyl on the beta-carbon atom linked to the 3-position on thepyridine ring; and D-cit refers to a D-isomer of citrulline.

Most preferably, the GnRH-2 antagonists are selected from the groupconsisting of peptides shown in SEQ ID NO: 10 to SEQ ID NO: 12. TheGnRH-2 antagonists shown in SEQ ID NOS: 10 to 12 are named Trptorelix.These amino acid sequences are illustrated below, and in the amino acidsequences of Trptorelix-2 and Trptorelix-3, amino acids different fromthe amino acid sequence of Trptorelix-1 are represented in bold Italics.

-   Trptorelix-1 (SEQ ID NO: 10):    Ac-D2Nal-(4Cl)D-Phe-D-3Pal-Ser-Tyr-D-Cit-Trp-Tyr-Pro-DAlaNH₂-   Trptorelix-2(SEQ ID NO: 11):    Ac-D2Nal-(4Cl)D-Phe-D-3Pal-Ser-His-DCit-Trp-Tyr-Pro-DAla-NH₂-   Trptorelix-3(SEQ ID NO: 12):    Ac-D2Nal-(4Cl)D-Phe-D-3Pal-Ser-Tyr-DCit-Trp-Leu-Pro-DAla-NH₂.

The GnRH-2 antagonists called Trptorelix are characterized by having Trpas their 7^(th) amino acid, and this is common to GnRH-2 and salmon GnRH(found only in fish, pyroGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-GlyNH₂ (SEQID NO: 14).

Trptorelix was constructed by modifying Cetrorelix (FIG. 1), awell-known antagonist of GnRH-1. Cetrorelix is an antagonist constructedby substituting the 1^(st), 2^(nd), 3^(rd), and 6^(th) amino acidsGnRH-1 with amino acid derivatives, and it shows a high binding affinitytoward GnRH-1 receptors but does not activate the receptors.Trptorelix-1 was synthesized by substituting the 7^(th) and 8^(th) aminoacids of Cetrorelix, which are very important in binding to the GnRH-1receptors, and thus this portion was changed to the amino acids ofGnRH-2 to increase the binding affinity toward the GnRH-2 receptors(FIG. 1).

Cell lines expressing the rat GnRH receptor, which very sensitivelyreacts to GnRH-1, and bfGnRHR, which readily reacts to GnRH-2, wereconstructed by the retrovirus-mediated infection method, and they weretreated with [D-Ala⁶]GnRH-2 and [D-Trp⁶]GnRH-2, which are GnRH-2agonists, and Trptorelix-1, which is a GnRH-2 antagonist, to investigatethe activity of the receptors. As a result, [D-Ala⁶]GnRH-2 increased theamount of inositol phosphate in a concentration-dependent manner in thecells expressing bfGnRHR and showed a 2.5˜10-fold higher sensitivitythan GnRH-2, and a 40˜20,000-fold higher sensitivity than GnRH-1 (FIG. 2a and FIG. 2 c). On the other hand, in the cells expressing the rat GnRHreceptor, [D-Ala⁶]GnRH-2 showed a 5-fold higher sensitivity than GnRH-2whereas it showed a 3.5-fold lower sensitivity than GnRH-1 (FIG. 2 d).These results show that [D-Ala⁶]GnRH-2 is a strong agonist specific toGnRH-2.

Also, in the cells expressing the Primate GnRH-2 receptor,[D-Ala⁶]GnRH-2 showed about a 3-fold excellent effect as compared withGnRH-2. On the other hand, it was revealed that [D-Lys⁶]GnRH-2 had abouta 1.5-fold lower effect than GnRH-2, and that [D-Trp⁶]GnRH-2 had a1.2-fold lower effect than GnRH-2. Accordingly, these results suggestthat with regard to the GnRH-2 receptors that are found in Primates andnon-mammals, [D-Ala⁶]GnRH-2 is the most effective agonist. Further, whenanalogs where the 5^(th), 7^(th), and 8^(th) amino acids of[D-Ala⁶]GnRH-2 are substituted were used, their effects were observed tobe weaker than [D-Ala⁶]GnRH-2, and it can therefore be seen that it isimportant to maintain His, Trp, and Tyr that are found at the 5^(th),7^(th), and 8^(th) positions of GnRH-2 as they are (FIG. 3 b).

[D-Ala⁶]GnRH-2 showed in vivo effects. When 10 μg of [D-Ala⁶]GnRH-2 wereinjected into a male rat, an increase in luteinizing hormone (LH) wasobserved in just one hour. This effect, however, was rapidly reducedafter 2 hours from the injection. On the other hand, testosterone, whichis a male hormone, began to rapidly increase from 2 hours after theinjection, it arrived at a peak point in 4 hours, and it exhibited asignificantly high value even after 8 hours as compared with before theinjection (FIG. 4).

The inventors revealed that a receptor with regard to GnRH-2 is presentin prostate cancer cells. When TSU-Pri cells, which are culturedprostate cancer cells, were treated with I¹⁵²-radiolabeled GnRH-1, nobinding with cells was observed, but when they were treated withI¹⁵²-radiolabeled GnRH-2, binding with cells was observed (FIG. 5 a andFIG. 5 b). In the latter case, when unradiolabeled [D-Ala⁶]GnRH-2 wastreated, the binding of I¹⁵²-radiolabeled GnRH-2 was reduced in aconcentration-dependent manner (FIG. 5 c). These results show that areceptor for GnRH-2 is present in prostate cancer cells and suggest that[D-Ala⁶]GnRH-2 can be used for the treatment of prostate cancer.

Trptorelix-1, which is an antagonist of GnRH-2, inhibits the productionof IP induced by GnRH-2 in a concentration-dependent manner, and itshows about a 100 to 2800-fold higher inhibitory effect on bullfrog GnRHreceptors than Cetrorelix, which is an agonist of GnRH-1 (FIG. 6 a andFIG. 6 b). On the other hand, Trptorelix-1 showed about a 60-fold lowereffect on rat GnRH receptors than did Cetrorelix (FIG. 6 c). Further,when cell lines expressing Primate GnRH-2 receptors were treated withTrptorelix-1 and Trptorelix-2, the activity of luciferase induced byGnRH-2 was reduced In a concentration-dependent manner, but treatmentwith Trptorelix-3 and Cetrorelix showed no effect (FIG. 7). It wasrevealed that, by substituting Leu and Arg which are the 7^(th) and8^(th) amino acids of Cetrorelix, with Trp and Tyr, Trptorelix showed ahigh binding affinity with regard to GnRH-2 receptors whereas it showeda low binding affinity with regard to GnRH-1 receptors. Such a resultshows that Trptorelix can effectively bind to GnRH-2 receptors andthereby inhibit their signal transduction. In summary, it is concludedthat Trptorelix is a very specific antagonist for GnRH-2.

The relative binding affinity of the agonists and antagonists toward thereceptors was determined, and as a result, the order ofTrptorelix-1>[D-Ala⁶]GnRH-2>GnRH-2>>[D-Ala⁶]GnRH-2>GnRH-1>Cetrorelixwith regard to the bfGnRH receptor was shown, and the order ofCetrorelix>Trptorelix-1>GnRH-1>[D-Ala⁶]GnRH-2>[D-Trp⁶]GnRH-2>GnRH-2 withregard to the rat GnRH receptor was shown (FIGS. 8 a and 8 b). Theseresults also show that [D-Ala⁶]GnRH-2 and Trptorelix-1 are an agonistand an antagonist specific to GnRH-2.

Also, [D-Arg⁶]GnRH-2, [D-leu⁶]GnRH-2, [D-Trp⁶]GnRH-2, and[D-t-bu-Ser⁶]GnRH-2, etc. described in PCT Laid-Open Publication No.01-74377 were subjected to experiments in cell lines having receptorssensitive to GnRH-1, but they were not studied with regard to receptorssensitive to GnRH-2. That is, the effect of these analogs on GnRH-2receptors was not verified. Particularly, the experiments conducted with[D-Trp⁶]GnRH-2 by the inventors revealed that [D-Trp⁶]GnRH-2 did nothave a significantly better effect than did a wild-type ligand, whereas[D-Ala⁶]GnRH-2 or [D-Lys⁶]GnRH-2 developed in the invention hadexcellent efficiency with regard to GnRH-2 receptors as compared withthe wild type ligand.

The present invention also relates to pharmaceutical compositions forregulating the release of gonadotropin comprising the agonists orantagonists regulating the activity of GnRH-2 by specifically binding tothe GnRH-2 receptors, and pharmacologically acceptable carriers.

The agonists and antagonists for GnRH-2 can be used for the treatment ofhuman reproductive physiological diseases and steroid-related cancercells, and they can also be used in the raising industry ofnon-mammalian animals, i.e., birds and fish. Accordingly, the GnRH-2agonists and antagonists can be employed for the prevention andtreatment of diseases associated with the activity of GnRH-2 byregulating the activity of GnRH-2.

The pharmaceutical compositions comprising the GnRH-2 agonists orantagonists can be suitably selected according to the purpose of use,the type of the GnRH-2 analogs to be used, the physical condition ofsubjects, and administration methods and forms. For example, the contentof the active ingredient of the pharmaceutical compositions may beprepared within the concentration of 0.1 nM-100 nM. The administrationmethods of the pharmaceutical compositions can be suitably selected by aperson having ordinary knowledge in the art to which the inventionpertains, and for example, oral administration or parenteraladministration can be employed. In an embodiment of the presentinvention, when the pharmaceutical compositions are prepared so that thecontent of the active ingredient can be at the concentration of 0.1nM-100 nM and then they are administered Into humans by injection, theamount by weight is about 0.1-100 μg/kg. Actually, in the invention,when a 100 g rat was treated with 10 μg of [D-Ala⁶]GnRH-2, it wasobserved that the concentration of luteinizing hormone and testosteronein blood was increased in vivo (FIG. 4). In an embodiment of the presentinvention, the GnRH-2 antagonists and agonists were first synthesized,and then after moisture was completely eliminated there from by coolingand concentrating them, they were stored in glass bottles in a vacuumstate. In the case of the agonists, they can be easily dissolved indistilled water, and the pharmaceutical compositions comprising them canbe prepared by diluting them in saline. In the case of antagonists, theycan be prepared by dissolving them in DMSO (dimethyl sulfoxide) ordiluting them with saline.

The GnRH-2 analogs can be used for treating steroid-related diseases(ex: prostate cancer, breast cancer, and ovarian cancer, etc.), diseasesassociated with the hypofunction of reproductive endocrinology (ex:irregular menstruation, amenorrhea, precocious puberty, andhypogonadism), the control of ovulation periods in in vitrofertilization (ex: the ovulation periods can be optionally controlled byadministering GnRH agonists or antagonists), contraception (ex: thegrowth of an embryo and implantation rate can be lowered by theadministration of GnRH antagonists after sexual intercourse), increasedproduction of fish and domestic animals, etc.

In connection with the molecular biological study of GnRH-2 receptorsand the assay of physiological functions of GnRH-2, all of the GnRHreceptors of non-mammals and the GnRH-2 receptor that has been found inmonkeys are more sensitive to GnRH-2 than GnRH-1 (Wang et al., ibid.;Neill et al., ibid.). Accordingly, the GnRH-2 agonists and antagonistscan be utilized for molecular biological study of these receptors, i.e.,the binding affinity of ligand-receptors and molecular study of signaltransduction mechanisms in cells. Further, although GnRH-2 is found inhumans and other mammals, its functions have not been known (White etal., ibid.). Hence, the GnRH-2 agonists and antagonists can be used tostudy the physiological functions of GnRH-2.

For the treatment of breast cancer, prostate cancer, and ovarian cancercells generated by steroids, GnRH-1 has been used (Schally, ibid. 1999;Grundker et al., ibid.). The agonists and antagonists of GnRH-1 havefunctions in regulating the secretion of steroids in the gonads, andthus, based on these functions, they have been used for the treatment ofthe cancers that are formed in prostate, breast, and ovary cells, aswell as cancer cells. That is, all of the strong agonists andantagonists of GnRH-2 inhibit the secretion of steroids in testes, andthereby the concentration of steroids in the body becomes low,ultimately causing the inhibition of the proliferation of cancer cells.

Recent studies have revealed that GnRH receptors are expressed in thesecancer cells, and thus studies about the direct effects of GnRH are nowin progress. In these cancer cells, GnRH-1 exhibited more effectivelythan GnRH-2. In other words, GnRH-2 inhibited the proliferation ofcancer cells at a lower concentration than that of GnRH-1 (Grundker etal., ibid.). Moreover, the inventors also proved that GnRH-2 bound moreeffectively to TSU-Pri cells, which are prostate cancer cells (FIG. 5).Therefore, when the analogs of GnRH-2 are used, they will inhibit theproliferation of cancer cells with a much lower cost because they can betreated at a lower concentration than that of the wild-type GnRH-2.Consequently, it is proposed that the agonists and antagonists of GnRH-2will be very effective for the treatment of steroid-related cancercells.

The agonists and antagonists of GnRH-2 can be used for the treatment ofreproductive endocrine diseases. Recent studies have revealed that, likeGnRH-1, GnRH-2 promoted the secretion of LH and FSH in the gonadal cellsof the pituitary gland (Millar et al., ibid.; Padmanabhan and McNeily,2001). While GnRH-1 is more effective for the secretion of LH, GnRH-2 ismore effective for the secretion of FSH. FSH and LH have functionsdifferent from each other in reproductive endocrinology. For example,FSH promotes the growth of follicles in ovaries, and LH helps therelease of ova from the matured follicles. It has been revealed that thesecretion function of FSH by GnRH-2 is stronger than the LH secretionfunction by GnRH-1. These studies suggest that the GnRH-2 agonists andantagonists can be used to regulate the secretion of FSH and LH. TheGnRH-1 has been used In hypogonadism, precocious puberty, irregularmenstruation, etc. thus far (Huirne and Lambalk, 2001). However, bothGnRH-1 and GnRH-2 promote the secretion of LH and FSH, but as they havedifferent characteristics in that the GnRH-1 promotes the secretion ofLH more effectively whereas the GnRH-2 promotes the secretion of FSHmore effectively, the mixed use of the GnRH-1 and GnRH-2 analogs willexhibit much better efficacy. Consequently, the agonists and antagonistsof GnRH-2 are expected to be able to be used for the treatment of suchreproductive endocrinology system diseases.

The agonists and antagonists of GnRH-2 can be used in in vitrofertilization. In infertility patients, in vitro fertilization is a veryimportant tool for pregnancy. For this purpose, the agonists andantagonists of GnRH are used in the control of ovulation periods.Likewise, the agonists and antagonists of GnRH-2 will be able to be usedfor the control of ovulation periods.

Also, the agonists and antagonists of GnRH-2 can be used forcontraception. The GnRH and GnRH receptors are expressed at the earlyembryo development stage. Particularly, when the GnRH was treated at theearly embryo development stage, the development of the embryo andimplantation were enhanced, whereas when the GnRH antagonists are used,the development of the embryo and implantation were inhibited (Raga F,et al., Endocrinology vol.140 pp. 3705-3712, 1999). Therefore, theagonists and antagonists of GnRH-2 will be able to be used forcontraception.

The agonists and antagonists of GnRH-2 can be used for the increasedproduction of fish and domestic animals. The GnRH receptors that havebeen found in fish and birds thus far are more sensitive to GnRH-2 thanGnRH-1. For over-ovulation of fish, GnRH-1 analogs have been used buttheir effects are insignificant. Consequently, [D-Ala⁶]GnRH-2 andTrptorelix, which act on the receptors that are sensitive to GnRH-2, canbe used for the increased production of fish and birds.

The present invention will be further described in more detail withreference to the following examples. The following examples are providedsolely to illustrate the invention: the protection scope of the presentinvention should not be construed to be limited thereto.

EXAMPLES Example 1 Construction of GnRH-2 Analogs

To develop agonists of GnRH-2, [D-Ala⁶]GnRH-2 in which Gly, the 6^(th)amino acid of GnRH-2, is substituted with D-Ala was constructed, and itssequence is shown in FIG. 1 and SEQ ID NO: 3. Also, [D-Ala⁶]GnRH-2 inwhich Gly at the 6^(th) position of GnRH-2 is substituted with D-Trp wasdesigned. The GnRH-2 was purchased from Sigma Co. (US), and its aminoacid sequence is shown in SEQ ID NO: 1.

Cetrorelix(Ac-D-2Nal-(4Cl)-D-Phe-D-3Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH₂), anantagonist of GnRH-1, was purchased from Serono Co. (Switzerland).Antagonists of GnRH-2 were constructed by substituting Tyr, Leu, andArg, which are the 5^(th), 7^(th), and 8^(th) amino acids of Cetrorelixwith His, Trp, and Tyr, and they were named Trptorelix-1, Trptorelix-2,and Trptorelix-3. They are shown in SEQ ID NOS: 10 to 12.

[D-Ala⁶]GnRH-2, [D-Trp⁶]GnRH-2, and Trptorelix were constructed bychemical synthesis by AnyGen Co. (located in Kwangju Institute ofScience and Technology, Kwangju, Republic of Korea).

Example 2 GnRH-2 Agonist-Induced GnRH-2 Receptor Activation

The GnRH receptor, a membrane protein connected with the G protein,activates the G protein when activated by a ligand, and the activated Gprotein activates phospholipase C and thereby the synthesis of inositolphosphate (IP), which is the second transmitter in cells, is increased.Based on this principle, the activity of a receptor can be investigatedby measuring the amount of IP within cells.

2-1 Inositol Phosphate Assay

GH3 cell lines were obtained from the Cell Line Bank at the CancerResearch Center, Seoul National University College of Medicine, locatedin Seoul, Republic of Korea. The GH3 cell lines were infected withretroviruses comprising genes encoding bfGnRHR-1, bfGnRHR-2, andbfGnRHR-3 which are bullfrog GnRH receptors, and rat GnRHR, and celllines expressing each of these receptors were obtained. The GH3 cellsexpressing bfGnRHR-1, bfGnRHR-2, bfGnRHR-3, and rat GnRHR were culturedon 12-well plates. After 24 hours, the cells were cultured again for 24hours in a media containing no inositol, to which H³-myoinositol wasadded. The resultant cells were washed with buffer A (140 mM NaCl, 20 mMHEPES, 4 mM KCl, 8 mM D-Glucose, 1 mM MgCl₂, 1 mM CaCl₂, 1 mg/ml BSA)and then cultured in buffer A containing 10 mM LiCl at 37°C. for 30minutes. Thereafter, they were treated with GnRH having a suitableconcentration, cultured at 37° C. for another 30 minutes, and then themedia was eliminated. The cells were treated with 10 mM of ice-coldformic acid to terminate the reaction, and the cell extracts were mixedwith Dowex anionic exchange resins. These resins were washed withdistilled water, IP was extracted with 1 M ammonium formate/0.1 M formicacid, and cpm (count per minute) was determined.

2-2: IP Production Induced by GnRH-2 Agonist

The GH3 cell lines were infected with retroviruses comprising genesencoding bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3 which are bullfrog GnRHreceptors, and rat GnRHR, and cell lines expressing each of thesereceptors were obtained. These cell lines were treated with GnRH-1,GnRH-2, and their agonists, and after 30 minutes, the formation of IP,which is the second signal transmitter, was investigated. In all thecell lines it was observed that when they were treated with GnRH and theagonists, IP was formed in a concentration-dependent manner. It wasobserved that with regard to all the bfGnRHR, GnRH-2 reacted moresensitively than GnRH-1, and with regard to the rat GnRH receptor,GnRH-1 reacted more sensitively than GnRH-2 (FIG. 2 a to FIG. 2 d, andTable 1).

These results agree with those that were already revealed, and theyconfirm that the non-mammalian GnRH receptors sensitively react toGnRH-2, and the mammalian GnRH receptor sensitively reacts to GnRH-1(Neill et al., ibid.; Millar et al., ibid.; Wang et al., ibid.). As canbe seen from FIG. 2 a to FIG. 2 d, when the GH3 cells expressingbfGnRHR-1 were treated with [D-Ala⁶]GnRH-2 and [D-Trp⁶]GnRH-2, which areagonists of GnRH-2, [D-Ala⁶]GnRH-2 activated bfGnRHR-1 at about an8-fold lower concentration than GnRH-2, and [D-Trp⁶]GnRH-2 activatedbfGnRHR-1 at a concentration similar to that of GnRH-2. When the GH3cells expressing bfGnRHR-2 were treated with GnRH-1, GnRH-2,[D-Ala⁶]GnRH-2, and [D-Trp⁶]GnRH-2, [D-Ala⁶]GnRH-2 acted on the receptormost sensitively and thereby induced the production of IP. GnRH-1 couldactivate bfGnRHR-2 at about a 1000-fold higher concentration thanGnRH-2. Moreover, it was observed that in the cell lines expressingbfGnRHR-3, [D-Ala⁶]GnRH-2 reacted most sensitively. On the other hand,in the cells expressing the rat GnRH receptor (rat GnRHR), GnRH-1induced the production of IP most sensitively and [D-Ala⁶]GnRH-2 andGnRH-2 showed a sensitivity lower than 10-fold.

TABLE 1 Log EC₅₀ Value for Each Receptor of GnRH GnRH-2 [D-Ala⁶]GnRH-2[D-Trp⁶]GnRH-2 GnRH-1 bfGnRHR-1 −9.41 ± 0.13 −9.91 ± 0.13 −9.40 ± 0.12−8.52 ± 0.14 bfGnRHR-2 −8.98 ± 0.14 −9.91 ± 0.19 −9.21 ± 0.13 −5.59 ±0.14 bfGnRHR-3 −9.51 ± 0.10 −9.89 ± 0.10 −9.22 ± 0.11 −8.42 ± 0.07ratGnRHR −8.06 ± 0.13 −8.49 ± 0.14 −7.92 ± 0.12 −9.69 ± 0.14

The above Table 1 shows the EC₅₀ value, the concentration inducinghalf-maximal stimulation of each receptor. The results as shown aboveexhibit that [D-Ala⁶]GnRH-2 very sensitively acts on bfGnRHR-1,bfGnRHR-2 and bfGnRHR-3, which are receptors that are sensitive toGnRH-2, whereas its sensitivity drops with regard to rat GnRHR, which issensitive to GnRH-1, and hence it is verified that [D-Ala⁶]GnRH-2 is anagonist specific to GnRH-2.

2-3:Luciferase Activity Assay

Complimentary DNA (cDNA) for a Primate GnRH-2 receptor was obtained froma CV-1 cell genome. A nucleotide sequence assay revealed that it showedthree nucleotide differences from the Primate GnRH-2 receptor that wasalready known (Neill et al., ibid.), and differences in two amino acidswere also found. Plasmids having Primate GnRH-2 receptors wheretransfected into CV-1 cells. To assay the activity of the receptor,c-fos-luc where a luciferase gene is fused with a c-fos promoter wasco-transfected as a probe gene. After 48 hours from transfection, thecells were treated with GnRH-2 agonists and then the activity ofluciferase was assayed (Seong et al., Endocrinology vol 144 2003,454-466).

2-4: GnRH-2 Agonist-induced Luciferase Activation

The injection of several GnRH-2 agonists into cells expressing PrimateGnRH-2 receptors revealed that they all increased the activation ofluciferase in a concentration-dependant manner. Of the GnRH-2 receptors,[D-Ala⁶]GnRH-2 performed excellently, showing effects about 3 times thatof a wild-type ligand, GnRH-2. In the case of [D-Lys⁶]GnRH-2 and[D-Trp⁶]GnRH-2, however, it was revealed that their effects were ratherreduced to about 1.5-fold and 1.2-fold, respectively, as compared withGnRH-2 (FIG. 3 a and Table 2). On the other hand, [D-Ala⁶]GnRH-1, whichis the agonist of GnRH-1, showed very low sensitivity as compared withGnRH-2. Accordingly, these results suggest that [D-Ala⁶]GnRH-2 is themost effective agonist for the GnRH-2 receptors that are found inPrimates and non-mammals. When analogs where the 5^(th), 7^(th), and8^(th) amino acids of [D-Ala⁶]GnRH-2 are substituted were used, it wasobserved that their effects were weaker than [D-Ala⁶]GnRH-2, andtherefore it can be seen that it is important to maintain His, Trp, andTyr that are found at the 5^(th), 7^(th), and 8^(th) positions of GnRH-2as they are (FIG. 3 b).

TABLE 2 Luciferase Activation by GnRH-2 Agonists in Cells ExpressingPrimate GnRH-2 Receptor E_(max) (fold- GnRH analogues Log EC₅₀ Ratio^(a)induction) GnRH-1 −5.74 ± 0.15 446.68 12.87 ± 1.12 GnRH-2 −8.39 ± 0.10 111.74 ± 0.31 [D-Ala⁶]GnRH-1 −6.38 ± 0.10 102.33  8.91 ± 0.39[D-Ala⁶]GnRH-2 −8.81 ± 0.12 0.38 11.84 ± 0.47 [D-Lys⁶]GnRH-2 −8.57 ±0.11 0.66 11.74 ± 0.43 [D-Trp⁶]GnRH-2 −8.31 ± 0.09 1.20 11.20 ± 0.32[D-Ala⁶, Leu⁷]GnRH-2 −7.95 ± 0.10 2.51  9.04 ± 0.27 [Leu⁵, D-Ala⁶]GnRH-2−7.35 ± 0.12 10.96  6.22 ± 0.25 [D-Ala⁶, Tyr⁷, −7.57 ± 0.13 6.61 12.83 ±0.56 Trp⁸]GnRH-2 [Tyr⁵, D-Ala⁶, −7.90 ± 0.24 3.01  7.35 ± 0.51Leu⁸]GnRH-2

Log EC₅₀ values and E_(max) values of GnRH-2 agonists are average valuesof experiments that were conducted independently three times. Ratio^(a)represents the ratio of EC₅₀ values of other GnRH analogs based on EC₅₀values of GnRH-2.

Example 3 GnRH-2 Agonist-Induced LH and Testosterone Secretion

Example 3: GnRH-2 Agonist-induced serum LH and testosterone levels[D-Ala⁶]GnRH-2 was dissolved in saline at a concentration of 100 μg/ml,and then 10 μg of [D-Ala⁶]GnRH-2 was injected into the cervix of a malerat (Sprague Dawley rat having a weight of approximately 100 g). 1, 2,4, and 8 hours after the injection, serum was obtained from the rat. Theconcentration of luteinizing hormone and testosterone present in theserum was measured by radioimmunoassay. When [D-Ala⁶]GnRH-2 was injectedinto the male rat, an increase of luteinizing hormone (LH) was observedin only one hour. This effect, however, was rapidly reduced by 2 hoursfrom the injection. On the other hand, testosterone, which is a malehormone, began to rapidly increase from 2 hours after the injection, itarrived at a peak point in 4 hours, and it exhibited a significantlyhigh value even after 8 hours as compared with before the injection(FIG. 4).

Example 4 Presence of GnRH-2 Receptor in Prostate Cancer Cells

To characterize the presence of GnRH-2 receptors in prostate cancercells, GnRH-1 and GnRH-2 were radiolabeled with I¹²⁵. TSU-Pri, which isone of the prostate cancer cell lines, was cultured along with theradiolabeled I¹²⁵GnRH-1 and I¹²⁵GnRH-2. As controls, the cellsexpressing rat GnRH receptors that readily bind to GnRH-1 and the cellsexpressing bfGnRHR-3 receptors that readily bind to GnRH-2 were used.The radiolabeled GnRH and the cells were cultured at room temperaturefor 6 hours, and then washed with PBS (phosphate-buffered saline)several times to eliminate the unbound GnRH. Thereafter, the cells wereisolated and radioactivity from the cells was measured using aγ-counter. I¹⁵²GnRH-1 bound to the cells expressing rat GnRH receptorsfairly well, and its binding was reduced when unradiolabeled GnRH-1 orGnRH-2 was treated as a competitor. On the other hand, when TSU-Pricells, which are prostate cancer cells, were treated withI¹⁵²-radiolabeled GnRH-1, no binding with cells was observed (FIG. 5 a).When TSU-Pri was treated with I¹⁵²GnRH-2, binding with cells wasobserved. In the latter case, when unradiolabeled GnRH-2 was used as acompetitor, the binding was reduced but when unradiolabeled GnRH-1 wasused as a competitor, the binding was not reduced (FIG. 5 b). Moreover,when unradiolabeled GnRH-2 or GnRH-2 agonists were used as competitorsat several concentrations, it was observed that the binding was reducedin a concentration-dependent manner (FIG. 5 c). These results show thata receptor for GnRH-2 is present in prostate cancer cells, and suggestthat [D-Ala⁶]GnRH-2 can be used for the treatment of prostate cancer.

Example 5 Effect of GnRH-2 Antagonists 5-1: Effect of GnRH-2 Antagonistson Non-Mammalian GnRH Receptor

When each GH3 cell expressing bfGnRHR-2 and bfGnRHR-3 constructed in 2-2above was treated with Trptorelix-1, which is the GnRH-2 antagonist,production of IP was not observed. However, Trptorelix-1 could reducethe production of IP induced by 1 nM GnRH-2, in aconcentration-dependent manner (FIG. 6 a to FIG. 6 c). Such effect wasrevealed to be 100-fold more sensitive with regard to bfGnRHR-2 andabout 1000-fold more sensitive with regard to bfGnRHR-3, than whentreated with Cetrorelix, which is the antagonist of GnRH-1 (FIG. 3)

TABLE 3 Log IC₅₀ Value for Each Receptor of GnRH Antagonist Trptorelix-1cetrorelix bfGnRHR-2 −8.10 ± 0.08 −6.43 ± 0.11 bfGnRHR-3 −8.49 ± 0.11−6.28 ± 0.13 ratGnRHR −9.27 ± 0.07 −11.03 ± 0.12 

On the other hand, Trptorelix-1 inhibited the activity of rat GnRHRinduced by GnRH-1 but it was shown that its sensitivity was very low ascompared with Cetrorelix, which is the antagonist of GnRH-1 (FIG. 6).Such a result suggests that Trptorelix specifically acts on bfGnRHR-3sensitive to GnRH-2 whereas its efficiency for the rat GnRHR sensitiveto GnRH-1 is lower than that of GnRH-1 antagonists. Table 3 shows theconcentration (IC₅₀) of antagonists inducing half-maximal inhibition ofGnRH receptor activity.

5-2: Effect of GnRH-2 Antagonists on Primate GnRH-2 Receptor

In the same manner as in 2-3 above, Primate GnRH-2 receptors wereco-transfected along with c-fos-luc reporter genes, and after 48 hours,GnRH-2 and GnRH-2 antagonists were treated thereinto at the same timeand then the effect of GnRH-2 antagonists was observed. When the cellline expressing the Primate GnRH-2 receptor was treated withTrptorelix-1 and Trptorelix-2, the activity of luciferase induced byGnRH-2 was reduced in a concentration-dependent manner, but Trptorelix-3and Cetrorelix showed no effect (FIG. 7 and Table 4). This shows that bysubstituting Leu and Arg, which are the 7^(th) and 8^(th) amino acids ofCetrorelix, with Trp and Tyr, Trptorelix showed a high binding affinitywith regard to GnRH-2 receptors whereas it showed low binding affinitywith regard to GnRH-1 receptors. Such result shows that Trptorelix caneffectively bind to GnRH-2 receptors and thereby inhibit their signaltransduction.

TABLE 4 Log IC₅₀ Value for Each Receptor of GnRH Antagonists. Reporterantagonist Log IC₅₀ c-fos-luc Trp-1 −5.74 ± 0.10 Trp-2 −6.19 ± 0.12Trp-3 ND^(a) Cetrorelix ND^(a)

Log IC₅₀ value by antagonists is an average value of experiments thatwere conducted independently three times. ND stands for “not detected”which means that there are no inhibitory functions.

Example 6 Ligand-Receptor Binding Assay

6-1

To determine the binding affinity between GnRH and GnRH receptors,GnRH-2 was radiolabeled with I¹²⁵. The GH3 cells expressing bfGnRHR-1,bfGnRHR-2, bfGnRHR-3, and rat GnRHR constructed in Example 2-3 abovewere cultured on 100-mm dishes. After 48 hours, the cells werehomogenized to obtain cellular membranes, which were then dissolved inbinding buffer (40 mM Tris, pH 7.4, 2 mM MgCl₂). 20 μg of membraneproteins were reacted with I¹²⁵-GnRH-2 at 4° C. for 16 hours. To thiswere added unradiolabeled GnRH-1, GnRH-2, [D-Ala⁶]GnRH-2,[D-Trp⁶]GnRH-2, and Trptorelix-1, which were then reacted along withI¹²⁵-GnRH-2 to investigate their binding affinity toward the receptors.Non-specific binding affinity was obtained from the competitive reactionwith 100 μM of GnRH-2. After the reaction was completed, 1 ¹²⁵-GnRH-2bound to the receptors was filtered using a Brandel harvester, and cpmwas measured using a γ-counter.

6-2: Binding Affinity of GnRH-2 Agonists and Antagonists towardReceptors

The GH3 cells expressing bfGnRHR-3 obtained from Example 6-1 above weretreated with l¹²⁵GnRH-2 and GnRH-2 agonists and antagonists at variousconcentrations to determine the binding affinity of GnRH-2 agonists andantagonists toward the receptors. With regard to bfGnRHR, the bindingaffinity was shown in the order ofTrptorelix-1>[D-Ala⁶]GnRH-2>GnRH-2>[D-Trp⁶]GnRH-2>GnRH-1>Cetrorelix, andwith regard to rat GnRHR, it was in the order ofCetrorelix>Trptorelix-1>GnRH-1>[D-Ala⁶]GnRH-2>[D-Trp⁶]GnRH-2>GnRH-2(FIG. 4 a to FIG. 4 b, and Table 5). These results suggest thatTrptorelix binds to bfGnRHR-3 more effectively than GnRH-2.

TABLE 5 Binding Affinity of GnRH Agonists and Antagonists towardReceptors (log IC₅₀ Value) GnRH-2 [D-Ala⁶]GnRH-2 [D-Trp⁶]GnRH-2 GnRH-1Cetrorelix Trptorelix-1 bfGnRHR-3 −8.63 ± 0.14 −9.83 ± 0.14 −8.10 ± 0.11−7.32 ± 0.17 −7.11 ± 0.08 −11.73 ± 0.19 ratGnRHR −6.08 ± 0.08 −6.85 ±0.13 −7.01 ± 0.09 −7.08 ± 0.10 −12.55 ± 0.15  −11.75 ± 0.19

The present invention provides agonists and antagonists ofgonadotropin-releasing hormone-2 (GnRH-2) which regulate the activity ofGnRH-2 by specifically binding to GnRH-2 receptors, and pharmaceuticalcompositions comprising them. Advantages thereof include theirusefulness for the treatment of reproductive physiology diseases andsteroid-related cancer cells because they specifically bind to theGnRH-2 receptors and effectively regulate GnRH-2, and they areapplicable to the raising industry of non-mammalian animals, i.e., birdsand fish.

1. A peptide-based gonadotropin-releasing hormone-2 (GnRH-2) antagonistwhich inhibits the activity of GnRH-2 by specifically binding to aGnRH-2 receptor, comprising the following peptide (SEQ ID NO:9):Ac-D-2Nal-(A)-D-Phe-D-3Pal-Ser-Xaa₅-D-Cit-Trp-Xaa₈-Pro-D-AlaNH₂; whereinA is 4C1, 4F, or 4 Br; Xaa₅ is Tyr or His; and Xaa₈ is Tyr or Leu. 2.The GnRH-2 antagonist of claim 1, wherein said antagonist is selectedfrom the group consisting of peptides shown in SEQ ID NO:10 to SEQ IDNO:12.
 3. A pharmaceutical composition for regulating the release ofgonadotropin comprising the GnRH-2 antagonist according to claims 1 or 2as an active ingredient, and a pharmaceutically acceptable carriers.